Introduction

Quillaja saponaria Molina (Quillay) is an endemic tree of four South American countries: Chile, Bolivia, Peru and Ecuador (^{INDAP, 2000}). In Chile its distribution bounded on the north by the parallel 30° 30’ south latitude, in the IV region of Coquimbo. The southern boundary is located north of the IX region around the city of Algol, 38° south latitude (^{Gallardo and Gastó, 1987}).

According to the Catastro y Evaluación de los Recursos Vegetacionales de Chile (Land Registry and Vegetational Resources Assessment of Chile) (^{CONAF et al., 1997}), Q. saponaria is part of the sclerophyllous forest type covering an area of 324 631 ha, equivalent to 2.6 % of the country’s native forests (^{Donoso and Escobar, 2006}). From its wood and bark, saponins are extracted which are economically very important molecules used in pharmaceutical, industrial and agricultural endings (^{Kensil et al., 1991}; ^{Hoffmann, 1995}). Currently, the high demand for its bark as the main source of saponins and land use change have reduced the presence of adult trees, particularly in the central area of Chile.

This situation was crucial to carry out a forestry and forest management project for the species, supported by a program of vegetative propagation (^{Prehn et al., 2003}), in which protocols of morphogenic development models were designed to propagate vegetatively and massively adult Quillay trees, using seeds as explants as they have a high content of saponins of good quality and low toxicity (^{Prehn et al., 2003}).

As an additional contribution to the in vitro propagation, in the present study a micropropagation technique known as direct organogenesis was tested. The effect of hormonal components on the morphogenic capacity of caulinary, apical and basal segments from adult trees were evaluated.

The practical utility of the method is to establish the differences and clarify aspects of the morphogenic competition from caulinary segments of adult individuals, which can be used as sources of germ plasm for clonal propagation.

Materials and Methods

The experiment was performed at the Laboratorio de Biotecnología Forestal (Laboratory of Forest Biotechnology) of the Universidad de Concepción, located in Concepción, Chile (36°46’ S and 73°03’ W), 500 km south of the capital. Concepción is a city and commune in south-central Chile, and is the capital of the VIII region of Bío Bío.

Experimental design and statistical analysis

The experiment was established under a completely balanced randomized design with five replications and factorial arrangement of treatments. The studied factors were the indole 3-butyric acid (IBA) and 6-benzylaminopurine (BAP) on three levels, for a total of nine treatments (Table 1). The experimental unit was a glass jar with four explants, which constituted 45 replicates per condition. After 90 days of culture, the number of buds, number of shoots and length of internodes per explant variables were evaluated. The information was subjected to an analysis of variance (ANOVA) through the PROC GLM SAS statistical program (^{Statistical Analysis System, 1999}). Comparison of treatment means was made with the multiple range test of Duncan procedure, with a significance level of 5 %.

Table 1 Treatments for of direct organogenesis induction in vitro in Quillaja saponaria Molina explants taken from different parts of the tree. 

Results and Discussion

In woody plants in vitro propagation is more difficult to achieve for its structural condition and maturity processes associated with age. In this regard, three processes that explain the above mentioned are described: chronological aging, based on changes expressed in function of time; ontogenetic aging, related to the gradual and irreversible transition regulated under genetic control; and physiological aging, associated with the loss of vigor caused by hormonal changes, nutritional and environmental conditions (^{Pierik, 1990}; ^{Russell et al., 1990}; ^{Gutiérrez, 1995}; ^{Pacheco, 1995}; ^{Andrés et al., 2002}). Specifically, the morphogenic capacity of the explant is strongly influenced by ontogeny, which varies according to the position of the plant material in the tree (^{Magini, 1984}).

In the present study greater morphogenic capacity explants from the basal area (Figure 1A) were observed, which is attributable to differences in the ontogeny of the tissue where buds originate, and by the various hormone levels in the different areas of the tree. These results agree with those obtained by ^{Fouret et al. (1986)}; ^{Thanh et al. (1987)} and ^{Yeung et al. (1998)}, who demonstrated the existence of greater competition in basal morphogenic microshoots and better use of nutritional reserves for cell elongation.

A, B and C. Proliferation of multi- growth zones; D, E and F. Elongation of microshoots from basal origin.

Figure 1. Photographic sequence exhibiting proliferation and elongation of the Quillaja saponaria Molina caulinary segments at 120 days of cultivation. 

^{Sánchez-Olate et al. (2005)} and ^{Hasbún (2006)} indicate that basal ontogenically microshoots are younger and show a better morphogenic response, because they have been generated from dormant meristem tissues. ^{Morisset et al. (2012)} point out the importance of other factors such as the amount of water, light and carbohydrates that basal microshoots used from the parent material during their development. The apical microshoots origin had lower rates of proliferation and close caulinary elongation. ^{Von Aderkas and Bonga (2000)}, ^{Andrés et al. (2002)}, ^{Fraga et al. (2002)}; ^{Tang et al. (2004)} and ^{Ramarosandratanam and Van Staden (2005)} suggest that differences in the responses of proliferation and elongation of the basal and apical microshoots could be linked to the existence of biochemical markers type and to differences in hormone levels within the donor tree.

According to the results of the ANOVA for the treatment factor, a significant effect (P <0.01, F = 77.46 and 12.82) was obtained on the variable of number of buds and for the length of internodes and buds. Specifically, for the variable number of buds T4 (1.0 / 2.0 mgL-1 of IBA / BAP) which originated a greater amount, with an average of seven shoots per explant (Table 2). In contrast, the T1 treatment (0.1 / 2.0 mgL-1 IBA / BAP) stimulated multiple points of growth (Figure 1A) and T5 (1.0 / 3.0 mgL-1 IBA / BAP) promoted buds with a rosette look and shape with plenty of surrounding callus (figures 1B and 1F).

Table 2 Comparative test between treatment means by Duncan’s multiple range test. 

Different letters mean statistical significant differences (P < 0.01).

Consistent with these results, it is demonstrated and substantiated the role of auxin-cytokinin hormone relations as a key factor in controlling morphogenesis. In addition, consistent with those obtained in Persea americana Mill by ^{Mohamed- Yasseen (1995)}; ^{Pliego-Alfaro and Murashige (1988)}, and in Persea lingue Ness by ^{Cob et al. (2010)}. Also, other studies agree that the organ induction effect of cytokinins is oriented to the formation of growth points, which reflected favorably with high proportions of cytokinin compared to auxin.

In several species it has been proved that as the concentration of cytokinin gets higher, a decrease in shoot length was found, until a rosette is formed (^{Sánchez-Olate et al., 2005}; ^{Rodríguez et al., 2003}; ^{Sotolongo et al., 2003}). In this regard, ^{Orellana (1998)} notes that the auxin-cytokinin balance is crucial for the coefficient of proliferation.

The length of the microstems is favored by decreasing the concentration of cytokinin in the IBA / BAP relationship; the optimum was reached with the T9 treatment (1.5 / 0.5 mg L-1 of IBA / BAP) (figures 1C, 1D, 1E). In contrast, the microshoots that were grown in T5 (1.0 / 3.0 mgL-1 IBA / BAP) did not elongate, and rosette-shaped explants were observed (Figure 1B).

Specifically, those in the T9 treatment reached, on average, 28 mm long and those of the T5 treatment, 12 mm average (Table 2).

These results coincide with those obtained in Juglans regia L. (^{Scaltsoyiannes et al., 1997}), Nerium oleander L. (^{Ollero et al., 2010}) and Legrandia concinna (Phil.) Kausel (^{Uribe and Cifuentes, 2004}). Authors show an increased cell elongation of lateral buds, by employing benzylaminopurine at low concentrations compared to auxin, attributable to decreased cell division and elongation of the tissue is promoted due to the action of auxin.

In this regard, ^{Von Arnold (1988)} notes that it is often necessary to reduce the dose of cytokinins in the nutrient medium to achieve an increase in the elongation of internodes, resulting in obtaining longest sprouts. ^{Guohua (1998)}, ^{Read and Preece (2003)} cite that cytokinins in high concentrations stimulate the proliferation of multiple growth points; in contrast, auxins induce the elongation of the cell wall, which is reflected in a lengthening of the microshoots. ^{Tamas (1995)} points out that the effect on caulinary elongation due to the addition and hormonal concentration in the culture medium is differential for each type of hormone which becomes evident when the auxin- cytokinin ratio levels vary.

Histological analysis included transverse and longitudinal cuts; it corroborated the in vitro regeneration process through direct organogenesis (figures 2A, 2B, 2C and 2D).

A. Longitudinal section of a protuberance separated from the initial tissue showing the presence of developing leaf primordia (PF) (bar = 5 mm); B. Longitudinal section of a protuberance with leaf primordia after 45 days from the start of the culture (bar = 5 mm); C. Cross section of a caulinary apex showing the formation of the meristematic dome (MD) and the presence of a meristemoide (M) (bar = 8 mm); D. Longitudinal section of a caulinary apex; note the presence of meristematic dome (DM) and meristemoids (M) (bar = 8 mm).

Figure 2 Histological sequence of formation of protuberances from nodal segments of Quillaja saponaria Molina cultured in vitro (40x). 

Figure 2A clearly shows a well-defined meristematic dome and leaf primordia. The meristematic dome is constituted by a layer of aligned surface cells, from which emerged the first pair of leaf primordia (figures 2B and 2C) that mark the beginning of the development of an adventitious vegetative shoot. Also, the processes involved in the formation of nodules became evident; it is observed that the main morphogenic activity of cells occurs in the tissues that make the meristematic region.

The results agree with those of ^{Mitra and Mukherjee (2001)} and ^{Lara et al. (2003)}, who point out that the cells of the meristematic zones are originating caulinary sprouts, through an organogenic process. These meristematic structures are characterized by always staying in a juvenile condition and are susceptible to external signals of induction and cellular reprogramming. Therefore, the formation of areas with morphogenic activity keep present in meristematic zones (Figure 2D), which by mitotic processes, first, form a meristematic dome and then the primordial leaves. This is attributable to high concentrations of hormones in the meristematic regions that promote active cell division and expansion permanently. Moreover, the exogenous application of growth regulators in the culture medium directly affected the endogenous hormone levels and, therefore, the meristematic activity that resulted in an increase of cellular development processes.

Conclusions

The in vitro culture of Quillaja saponaria is successful in the stages of multiplication and elongation of caulinary microshoots explants from the basal area. Specifically, by applying an optimal ratio of indol3-butyric acid (IBA) and 6-benzylaminopurine (BAP) in the nutrient medium, the development of adventitious organogenesis was stimulated, which is corroborated by histological analysis. On the other hand, the contribution of this study lies in affirming the scientific foundation around the morphogenic competition explants from different heights of the donor tree. However, there are still questions on the subject, worthy of being incorporated in future studies. It is also required to optimize processes based on the replication of other assays under similar conditions.